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Notes: To better understand the role MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), in reducing retrotransposon activity, 293T human embryonic kidney cells expressing MOV10 and one of three retrotransposons (LINE1 (L1), Alu or SVA) were lysed in a buffer that included RNasin® Ribonuclease Inhibitor, and FLAG-tagged L1 complexes immunoprecipitated and analyzed by mass spectrometry. Several retrotransposon assays were conducted using FuGENE® HD Transfection Reagent for transfected constructs into 293T, HeLa and HeLa-HA cells. (4255)

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

Notes: The authors investigated the role of estrogen inactivation by the SULT1E1-encoded estrogen sulfotransferase in ovulation in mice. Semiquantitative RT-PCR was used to characterize the temporal and tissue-specific expression of SULT1E1 mRNA in ovulating mice. First-strand cDNA synthesis was performed at 37°C for 2 hours using 7.5µg of total RNA, 1µl (0.5µg) of oligo(dT)15, 40 units of RNasin® Ribonuclease Inhibitor and 200 units of M-MLV Reverse Transcriptase. (3913)

Notes: The authors screened members of a class of acylated hydrazones of salicylaldehydes (INPs) to characterize their ability to inhibit growth of Chlamydia by affecting the type III secretion (T3S) system, a potent virulence mechanism. Expression levels of various T3S genes and gene markers of early, middle and late developmental cycles were examined by RT-PCR in Chlamydia trachomatis-infected HeLa 229 cells in the presence and absence of INPs. HeLa229 RNA was isolated at 4, 8, 24 and 36 hours postinfection, treated with RQ1 RNase-Free DNase and amplified using the Access RT-PCR System in the presence of 0.5 units of RNasin RNase Inhibitor. Each set of experiments included a no-reverse transcriptase control reaction to control for DNA contamination and a positive control reaction using the Positive Control RNA with Carrier and control primers supplied with the kit. (3795)

Notes: Human arsenic methyltransferase (AS3MT) was cloned and mutated to produce allozymes for further analysis. COS-1 cells were transfected with constructs encoding the wildtype enzyme and the synthetic allozymes using TransFast™ Transfection Reagent. The wildtype enzyme and allozymes were transcribed and translated using TNT® T7 Coupled Reticulocyte Lysate System in the presence of RNasin® Ribonuclease Inhibitor. (3383)

Notes: MCF-7 cells were incubated for 24 hours in PRF-SFM and then detached and plated on uncoated dishes or dishes coated with 2μg/cm2 poly-L-Lysine (P-Lys) in PBS, 30μg/ml fibronectin (Fn) in PBS or 30μg/ml type IV Collagen (Col) in 10mM acetic acid. The cells plated on uncoated dishes were treated both with and without 10nM estradiol (E2). After 24 hours, total cellular RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Briefly, reverse transcription was performed on 1μg of total RNA in a final volume of 10μl by incubation at 37°C for 30 min with 200U of M-MLV reverse transcriptase, 0.4μg oligo-dT, 0.5μM dNTP and 24U RNasin® Ribonuclease Inhibitor, followed by heat denaturation for 5 minutes at 95°C. Subsequent PCR analysis was performed on 1μl of the RT product in a final volume of 25μl. Primers were used to amplify the 210bp fragment of PS2 and the 304 bp fragment of cathepsin D. Amplification of 408bp of ribosomal RNA 36B4 was performed as control. The PCR mixture consisted of 1.25U GoTaq® DNA Polymerase, 1X PCR buffer (10mM Tris-HCl, 50mM KCl), 2.5mM MgCl2, 0.2mM each dNTP, 0.6μM of each PS2 primer or cathepsin D primer and 0.2μM of each 36B4 primer. PCR was performed for 20 cycles at 95°C for 1 minute, 59°C for 2 minutes and 72°C for 1 minute. Ten microliters of the PCR products were separated on a 1.2% agarose gel. (3377)

Notes: RNA was extracted from 300mg malignant mesothelioma tissue samples and treated with DNase in the presence of RNasin® Ribonuclease Inhibitor. The RNA was incubated with oligo(dT) primers and reverse transcribed in the presence of radiolabeled nucleotides. The labeled cDNA was hybridized to preprinted microarrays containing 4132 human genes. (2715)

Notes: The patch-clamp technique was combined with RT-PCR in acute hippocampal brain slices. After recording, each cell was harvested to perform transcript analysis and identify subunits that underlie inwardly rectifying K+ currents. The recording pipette solution was supplemented with recombinant RNasin Ribonuclease Inhibitor. After recording channel activity, cell contents were harvested through the recording pipette. RT-PCR was performed on cell contents. (2426)

Notes: The authors describe using the Wizard® Genomic DNA Purification kit and the SV Total RNA Isolation System to isolate genomic DNA and total RNA, respectively, from Streptococcus pneumoniae. The researchers also added RNasin® Ribonuclease Inhibitor to purified total RNA before storing it and using it for later RT-PCR reactions performed with the Access RT-PCR System. (2835)

J. Bacteriol.183, 7094-7101.
Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions.2001

Vandecasteele, S.J., Peetermans, W.E., Merckx, R. and Van Eldere, J.

Notes: M-MLV Reverse Transcriptase and RNasin® Ribonuclease Inhibitor were used in a reaction to generate first strand cDNA. The generated cDNA was used as a template for quantitative PCR in a Taqman® Assay. The pGEM®-T Vector System was used to clone copies of each target gene so that standards could be generated. The Wizard® Genomic DNA Purification Kit was used to isolated genomic DNA from the S. epidermidis to generate a standard curve for quantitation of genomic DNA contamination of samples. (2291)

Brain Res. Mol. Brain Res.76, 25–35.
Expression of the GDNF family members and their receptors in the mature rat cochlea.2000

Stöver, T., Gong, T.L., Cho, Y., Altschuler, R.A. and Lomax, M.I.

Notes: Total RNA was isolated from various rat tissues with the SV Total RNA Isolation System. Yields are reported as 10µg from 16 whole cochlea, 8µg from 16 modiola, 10.4µg from 48 cochlear sensorineural epithelial/lateral walls and 50µg from the substantia nigra region of four brains. The isolated RNA was used for RT-PCR in the presence of RNasin® Ribonuclease Inhibitor. The resulting amplimer was subcloned into the pGEM®-T Easy Vector and clones were purified with the Wizard® Plus SV Minipreps System. (2176)

Notes: Reverse transcription reaction was performed in the presence of 25ug/ul Single-Stranded DNA Binding Protein and 1 unit/ul RNasin® Ribonuclease Inhibitor. PCR product and 35S-methionine were added to the TNT® Coupled Reticulocyte Lysate System to produce labeled protein in a PTT assay. (1185)

Notes: Genomic DNA was extracted from 100 Drosophila melanogaster and 100 Drosophila virilis using the Wizard® Genomic DNA Purification Kit. The isolated genomic DNA was used for PCR amplification and direct sequencing. RT-PCR was also performed in the presence of RNasin® Ribonuclease Inhibitor and cloned amplimers were purified with the Wizard®Plus Minipreps DNA Purification System prior to fluorescent sequencing. (2505)

Notes: The factors BDNF and NT-3 were used to maintain cultures. RNasin® Ribonuclease Inhibitor and Taq DNA Polymerase were used for RT-PCR of rat total RNA derived from arcuate and periventricular cultures from newborn rats as well as intermediate lobe cells from adult rats. (0745)

Notes: Genomic DNA was isolated from mouse brain with the Wizard® Genomic DNA Purification Kit. The isolated DNA was used for PCR. The RNasin® Ribonuclease Inhibitor was used to protect RNA in RT-PCR. (0372)

Notes: A novel furin-like human articular cartilage, cartilage intermediate layer protein (CILP), was expressed in the TNT® T3 Coupled Reticulocyte Lysate System. The CILP cDNA was cloned into a pBluescript II KS(1) vector. The researchers used 500ng of the plasmid in the in vitro transcription/translation reaction. Reactions were supplemented with [35S]methionine and 20 units RNasin® Ribonuclease Inhibitor. The reaction products were ethanol precipitated and resuspended in SDS loading buffer before loading on a SDS-PAGE gel for visualization. (2687)

Notes: The Core Footprinting System was used to footprint a region of the CYP2A3 gene with nuclear extracts derived from various rat and mouse tissues. The RNasin® Ribonuclease Inhibitor was used to protect transcripts of a eukaryotic in vitro transcription reaction. The transcripts were used in an S1 Nuclease protection assay. (0091)

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